Seo Jin Kim

Analysis of gene expression profiles in cholesteatoma using oligonucleotide microarray.

Abstract Conclusion. Microarray analysis may be a useful tool to identify some candidate genes related to the pathogenesis of cholesteatoma. Objective. The aim of this study was to investigate gene expression profiles in human cholesteatoma using an oligonucleotide chip including 10115 genes. Materials and methods. Gene expression from five cholesteatoma matrices and five normal retroauricular skins was analyzed by Macrogen human oligo-chip and the expression levels of some selected genes were also confirmed by RT-PCR. Results. In all, 1327 up-regulated or 767 down-regulated genes that were over 3 times more prominent in cholesteatoma than in skin were identified by 5 samples of microarray data. Among these up-regulated or down-regulated genes in cholesteatoma, 291 genes were identified in 3 samples or more out of 5 samples as up-regulated expression more than threefold in density and 191 genes were down-regulated more than threefold in density. RT-PCR of 21 selected genes revealed that those expression levels were higher in choleasteatoma than retroauricular skin.

Differential gene expression profiles in salicylate ototoxicity of the mouse

CONCLUSION This study demonstrated differential gene expression profiles in salicylate ototoxicity with oligonucleotide microarray. This study may also provide basic information on candidate genes associated with hearing loss and/or tinnitus or recovery after salicylate-induced cochlear dysfunction. OBJECTIVES Salicylate ototoxicity is accompanied by temporary hearing loss and tinnitus. The purpose of the present study was to evaluate the gene expression profiles in the mouse cochlea with salicylate ototoxicity using DNA microarray. MATERIALS AND METHODS The subject mice were injected intraperitoneally with 400 mg/kg of sodium salicylate; an approximate 30 dB threshold shift that was observed by auditory brainstem response was achieved 3 h after an injection of sodium salicylate and the hearing threshold returned to within normal range at 3 days. Differential gene expression profiles at 3 h after salicylate injection in comparison to the normal cochlea were analyzed with DNA microarray technology. RESULTS No ultrastructural changes in the mice cochlea were observed by TEM at 3 h after salicylate injection. Microarray revealed that 87 genes were up-regulated twofold or more in the mouse cochlea with salicylate ototoxicity in comparison to the normal cochlea. Among these genes, increased expression levels of 30 functional genes were confirmed by semi-quantitative RT-PCR.Conclusion. This study demonstrated differential gene expression profiles in salicylate ototoxicity with oligonucleotide microarray. This study may also provide basic information on candidate genes associated with hearing loss and/or tinnitus or recovery after salicylate-induced cochlear dysfunction. Objectives: Salicylate ototoxicity is accompanied by temporary hearing loss and tinnitus. The purpose of the present study was to evaluate the gene expression profiles in the mouse cochlea with salicylate ototoxicity using DNA microarray. Materials and methods: The subject mice were injected intraperitoneally with 400 mg/kg of sodium salicylate; an approximate 30 dB threshold shift that was observed by auditory brainstem response was achieved 3 h after an injection of sodium salicylate and the hearing threshold returned to within normal range at 3 days. Differential gene expression profiles at 3 h after salicylate injection in comparison to the normal cochlea were analyzed with DNA microarray technology. Results: No ultrastructural changes in the mice cochlea were observed by TEM at 3 h after salicylate injection. Microarray revealed that 87 genes were up-regulated twofold or more in the mouse cochlea with salicylate ototoxicity in comparison to the normal cochlea. Among these genes, increased expression levels of 30 functional genes were confirmed by semi-quantitative RT-PCR.

Do microRNA 96, 145 and 221 expressions really aid in the prognosis of prostate carcinoma?

MicroRNAs (miRs) are small noncoding RNAs that have been reported to be promising diagnostic tools. We used quantitative real-time reverse transcription PCR (RT-qPCR) to analyze differentially expressed miRNAs in prostate tumor samples to determine its prognostic value. From 2007 to 2009, tumor tissues were obtained from 73 radical prostatectomy specimens. Differentially expressed miR-96, -145 and -221 were validated by TaqMan RT-qPCR using all 73 tissues. The prognostic value was assessed in terms of biochemical recurrence using Kaplan-Meier and Cox regression analyses. For our patient cohort, the mean age was 64.7 years (50-76 years) and the mean prostate-specific antigen (PSA) was 7.5 ng ml(-1). During the follow-up period (mean, 19.4 months), 14 of 73 (19.2%) patients developed biochemical recurrence. Expression of miR-96, -145 and -221 correlated strongly with each other, but there were no correlations between miRNA expression and clinicopathologic parameters. Kaplan-Meier survival curves using the log-rank test showed a decreased biochemical recurrence-free interval with pathologic stage (P<0.001). In addition, patients with Gleason scores over 8, compared with those with a Gleason score of 6, showed a decreased biochemical recurrence-free interval in Kaplan-Meier analysis (P=0.001). However, expression of miR-96, -145 and -221 did not correlate with the biochemical recurrence interval in Kaplan-Meier survival curves or by multivariate analysis using the Cox proportional hazard regression model, either. In conclusion, we did not observe a significant correlation between the expression of miR-96, -145 and -221 and clinicopathologic parameters. To utilize miRNA as a diagnostic tool in clinical practice, more research is needed to understand miRNA mechanisms, identify miRNA targets, and further characterize miRNA function.

Up-regulation of MUC5AC mRNA expression in endotoxin-induced otitis media

We investigated the expression levels of MUC5AC in endotoxin-induced otitis media with effusion (OME) in the rat using competitive polymerase chain reaction (PCR) and the morphology of middle ear mucosa using transmission electron microscopy (TEM). Experimental OME in the rat was induced after middle ear instillation of Escherichia coli lipopolysaccharides (LPS). Middle ear mucosa were obtained at 0 h, 12 h, Day 1, Day 3, Day 7 and Day 14 and reverse transcriptase (RT)-PCRs were then performed for the identification of MUC1, MUC2, MUC5AC and submandibular mucin 1 expression, followed by competitive PCRs for MUC5AC and beta2-microglobulin expression. Normal middle ear mucosa revealed no expression of mucin genes, whereas endotoxin upregulated the expression of MUC5AC mRNA between 12 h and Day 7, with maximal expression at Days 1 and 3. Middle ears treated three times with LPS upregulated more MUC5AC mRNA expression, by a factor of approximately 3.5, than those 1 day after one instillation. On TEM, dark granulated cells were observed at Day 3 after endotoxin instillation, but mixed granulated cells were seen on the ears treated three times with LPS. These results suggest that MUC5AC could be one of the major mucin genes in the middle ear mucosa related to otitis media.We investigated the expression levels of MUC5AC in endotoxin-induced otitis media with effusion (OME) in the rat using competitive polymerase chain reaction (PCR) and the morphology of middle ear mucosa using transmission electron microscopy (TEM). Experimental OME in the rat was induced after middle ear instillation of Escherichia coli lipopolysaccharides (LPS). Middle ear mucosa were obtained at 0 h, 12 h, Day 1, Day 3, Day 7 and Day 14 and reverse transcriptase (RT)-PCRs were then performed for the identification of MUC1, MUC2, MUC5AC and submandibular mucin 1 expression, followed by competitive PCRs for MUC5AC

Expression of vascular endothelial growth factor receptors in experimental otitis media in the rat

Objective --Increased vascular permeability and endothelial cell growth are important in the pathogenesis of otitis media with effusion (OME) and vascular endothelial growth factor (VEGF) is known to play an important role in the increased vascular permeability and angiogenesis associated with OME. The action of VEGF is mediated by two different high-affinity receptors–VEGF receptor-1 (VEGFR-1; flm-like tyrosine kinase-1; flt-1) and VEGFR-2 (kinase insert domain-containing receptor; flk-1)–predominantly located on the vascular endothelium. The purpose of this study was to investigate the expression of three forms of VEGFR (-1, -2 and -3) in an endotoxin-induced rat model of OME. Material and Methods --Middle ear mucosa were obtained at 0, 1, 3, 6 and 12 h and 1, 3, 7 and 14 days after induction, and the expression of VEGFR mRNA and protein was evaluated using semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting. Results --RT-PCR revealed that the expression of VEGFR-1 and -2 mRNA was upregulated between 1 h and 3 days after endotoxin instillation, with peak expression occurring at 12 h and on Day 1. No expression of VEGFR-3 mRNA was detected in any of the samples. Expression patterns of VEGFR-1 and -2 protein observed by Western blotting were similar to those of VEGFR-1 and -2 mRNA and peak expression was observed on Day 1. Expression of VEGFR mRNA or protein was not detected in either normal or saline-instilled middle ear mucosa. Conclusion --These results suggest that both VEGFR-1 and -2 are upregulated during experimental otitis media in the rat and these receptors may play different roles in the production of effusion in OME.

Expression of β-defensins in the tubotympanum of experimental otitis media

Conclusion. Since the expression levels of β-defensins 2–4 were up-regulated in experimental otitis media, they may play a protective role in the pathogenesis of otitis media. Objectives. Defensins are antimicrobial peptides that play a major role in innate immunity. The goal of this study was to identify the expression of defensins in experimental otitis media of the mouse. Materials and methods. The expression of three α-defensins (cryptdins, cryptdin-related sequences 1-C (CRS1-C), and CRS4-C) and four β-defensins (mBD1, mBD2, mBD3, and mBD4) was investigated in the tubotympanum of experimental otitis media in mice by a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), and the expression levels of three β-defensins (mBD2, mBD3, mBD4) were evaluated by Western blotting. Results. The α-defensins were not expressed in the tubotympanum. mBD1 was expressed constitutively in normal middle ear mucosa and Eustachian tube mucosa, but up-regulated expression of mBD2, mBD3, and mBD4 was observed with RT-PCR and Western blotting in the tubotympanums in experimental otitis media, while the normal tubotympanums did not express them.

Expression of Insulin-like Growth Factors in a Mouse Model of Salicylate Ototoxicity.

Objectives Insulin-like growth factors (IGFs) are known to be neurotrophic factors, and they efficiently signal to cells to grow, differentiate and survive. The purpose of study was to identify the expressions of IGFs in mice with salicylate ototoxicity, which is a typical reversible hearing loss model. Methods The mice were given intraperitoneal injections of salicylate (400 mg/kg) and about a 30 dB threshold shift was achieved at 3 hours. The expressions of IGF-1 and 2 were confirmed by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blotting. Localization of IGFs was confirmed using confocal immunofluorescence imaging. For in-vitro study on the HEI-OC1 auditory cells, the cell viability was calculated and the apoptotic features of the nuclei were observed with Hoechst staining. Results The expressions of the IGFs mRNA and protein were significantly increased in the salicylate ototoxicity groups compared with that of the normal control group. Salicylate induced apoptosis and decreased viability of the HEI-OC1 auditory cells in a time- and dose-dependent manner. The expressions of IGFs were localized in the stria vascularis, and these IGFs play a protective role in the in-vivo condition of salicylate ototoxicity. Conclusion IGFs were highly expressed in the mice with salicylate ototoxicity, and this expression was mainly focused in the stria vascularis in the salicylate intoxicated mice. The systemic action of IGFs, which were expressed in the vascular-rich stria vascularis, can act as a major protective mechanism in a mouse model of salicylate ototoxicity.

Expression of Clara cell secretory protein in experimental otitis media in the rat.

Conclusion These results suggest that CCSP is upregulated in OME and may play a protective role in the pathogenesis of OME. Objective Clara cell secretory protein (CCSP) is an abundant 16-kDa homodimeric protein and is secreted by non-ciliated secretory epithelial cells in the lung. It has an important protective role against the intrapulmonary inflammatory process. The aim of this study was to investigate the expression of CCSP in endotoxin-induced otitis media with effusion (OME) in the rat. Material and methods We instilled endotoxin and saline (control) into the middle ear cavity of the rat. Middle ear mucosa were taken at 0, 1, 3, 6 and 12 h and 1, 3, 7 and 14 days, and the expression of both CCSP mRNA and protein were then evaluated using semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR), Western blotting and immunohistochemistry. Results RT-PCR revealed that the expression of CCSP was first identified at 1 h after endotoxin instillation, was dramatically increased between 1 h and Day 1, with maximal expression at 12 h, and then decreased after Day 3. The expression pattern of CCSP protein identified by means of Western blotting was similar to the CCSP mRNA patterns observed using RT-PCR. Expression of CCSP at both mRNA and protein levels was not detected in either normal middle ear mucosa or saline-instilled middle ear mucosa. Immunohistochemistry revealed that some epithelial cells in the middle ear mucosa were stained.

Gene expression profiles of rat olfactory bulb at developmental stage

Microarray analysis may be a useful tool to identify some candidate genes related to the development of olfactory bulbs. In the present study, gene expression profiles of olfactory bulbs from postnatal day 1 (P1) rats and postnatal day 35 (P35) rats were analyzed by oligonucleotide-microarray and expression levels of some selected genes were also confirmed by RT-PCR and in situ hybridization. 9146 genes were commonly identified in six microarray chips. Among these genes, 76 were up-regulated and 130 were down-regulated three-folds or more at P1 olfactory bulbs. Out of these 76 up-regulated genes, 24 genes were annotated based on the NCBI database of reference sequences and expression levels of these 24 genes were confirmed by RT-PCR. Among them, 2 interesting genes (neurogenic differentiation 1 and retinoid acid receptor alpha) were localized in the P1 olfactory bulb by the use of in situ hybridization technique. Our results may provide basic information to identify genes associated with functional growth of olfactory bulbs.

Expression of the Acidic Fibroblast Growth Factor and Basic Fibroblast Growth Factor in Nasal Polyps

CONCLUSION Up-regulated expression of acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor (bFGF) in nasal polyps may be related to epithelial proliferation and neoangiogenesis associated with nasal polyp growth. BACKGROUND AND OBJECTIVES aFGF and bFGF are polypeptides that effect mitogenesis, neoangiogenesis and tissue repair. The purpose of this study was to investigate the expression of aFGF and bFGF at both the mRNA and protein levels in nasal polyps. MATERIALS AND METHODS Tissue samples of nasal polyp and inferior turbinate mucosae were obtained. Reverse transcriptase (RT)-PCR, in situ hybridization, and Western blotting for aFGF and bFGF were performed in nasal polyp and nasal inferior turbinate. RESULTS RT-PCR showed 50% aFGF and 29% bFGF expression positivity in 14 nasal polyps; however, 13 turbinates revealed no PCR products. aFGF and bFGF expression was localized to some inflammatory cells of nasal polyps by in situ hybridization. Western blotting identified aFGF and bFGF molecular masses of 18 kDa and 24 kDa, respectively, in nasal polyps, and these levels were higher than those observed in nasal turbinates. aFGF and bFGF proteins were also localized in some inflammatory cells of nasal polyps by immunostaining.